Project Summary/Abstract Compared with individuals expressing the most common genetic variant of apolipoprotein E (apoE), apoE3, those harboring apoE4 are at increased risk of both cardiovascular disease and type 2 diabetes mellitus (T2DM). Although apoE classically participates in lipid transport, the basis for apoE4-associated risk remains unclear, and there are no interventions available to break the link between apoE4 and vascular and metabolic disorders. We recently discovered that the insulin resistance observed in mice expressing human apoE4 is driven by skeletal muscle insulin resistance mediated by the apoE receptor apoER2 in endothelial cells. Studies in culture showed that the underlying mechanism is apoE4-apoER2-induced inhibition of endothelial cell insulin transport, which has a major impact on insulin action in muscle. In other recent work we determined that thrombosis is markedly more severe in apoE4- versus apoE3-expressing mice. Cell culture studies further indicated that this may be due to apoE4 promotion of von Willebrand factor (vWF) secretion by endothelial cells. The Overall Goal of the proposed research is to elucidate how apoE4 actions on endothelium influence glucose homeostasis and vascular disease pathogenesis. Aim 1 will determine how apoE4 causes insulin resistance. A non-biased query of apoER2-interacting proteins and ensuing studies of insulin transport in cultured endothelial cells suggest involvement of Dab2-interacting protein (DAB2IP) and JIP1, which both modulate JNK signaling. Using endothelial cell-specific deletion in apoE3- and apoE4- expressing mice, we will test the hypothesis that apoE4 promotes insulin resistance via endothelial apoER2 through DAB2IP- and JIP1-dependent mechanisms. Aim 2 will determine how apoE4 promotes atherosclerosis and thrombosis. Studies of proteins interacting with apoER2 in endothelium and apoE4 enhancement of endothelial cell vWF secretion and monocyte adhesion indicate possible participation of the protein phosphatase PP2A. In apoE3- and apoE4-expressing mice we will test the hypothesis that apoE4 promotes atherosclerosis and thrombosis via endothelial apoER2-related activation of PP2A. In Aims 1 and 2, we will additionally use GWAS-based strategies to determine if the operative genes in mice are modifier genes influencing apoE4-related risk of metabolic and vascular disease in humans. In Aim 3 we will devise interventions against apoE4 and its mechanisms of action to preserve metabolic and vascular health. In apoE4 mice the effect of JNK inhibition on insulin resistance and the effect of PP2A inhibition on atherosclerosis and thrombosis will be evaluated. We will also use base editing to genetically correct apoE4 to apoE3 in vivo in the hepatocytes of apoE4 mice. Editing efficiency and impact on apoE4-related insulin resistance, atherosclerosis and thrombosis will be evaluated. By accomplishing these Aims and unraveling how apoE4 promotes cardiometabolic disease, it is expected that novel therapies can then be developed to neutralize an important genetic contribution to metabolic and vascular disease in 15 to 20% of the population.